Daniel Kalderon

Hedgehog acts as a somatic stem cell factor in the Drosophila ovary.

Secreted signalling molecules of the Hedgehog (Hh) family have many essential patterning roles during development of diverse organisms including Drosophila and humans. Although Hedgehog proteins most commonly affect cell fate, they can also stimulate cell proliferation. In humans several distinctive cancers, including basal-cell carcinoma, result from mutations that aberrantly activate Hh signal transduction. In Drosophila, Hh directly stimulates proliferation of ovarian somatic cells. Here we show that Hh acts specifically on stem cells in the Drosophila ovary. These cells cannot proliferate as stem cells in the absence of Hh signalling, whereas excessive Hh signalling produces supernumerary stem cells. We deduce that Hh is a stem-cell factor and suggest that human cancers due to excessive Hh signalling might result from aberrant expansion of stem cell pools.

Proteolysis of the Hedgehog Signaling Effector Cubitus interruptus Requires Phosphorylation by Glycogen Synthase Kinase 3 and Casein Kinase 1

The secreted signaling molecule Hedgehog regulates gene expression in target cells in part by preventing proteolysis of the full-length Cubitus interruptus (Ci-155) transcriptional activator to the Ci-75 repressor form. Ci-155 proteolysis depends on phosphorylation at three sites by Protein Kinase A (PKA). We show that these phosphoserines prime further phosphorylation at adjacent Glycogen Synthase Kinase 3 (GSK3) and Casein Kinase I (CK1) sites. Alteration of the GSK3 or CK1 sites prevents Ci-155 proteolysis and activates Ci in the absence of Hedgehog. Ci-155 proteolysis is also inhibited if cells lack activity of the Drosophila GSK3, Shaggy, previously implicated in Wingless signaling. Conversely, Ci-155 levels are reduced in Hedgehog-responding cells by overexpression of PKA and the Drosophila CK1, Double-time, a regulator of circadian rhythms.

FUNCTION OF PROTEIN KINASE A IN HEDGEHOG SIGNAL TRANSDUCTION AND DROSOPHILA IMAGINAL DISC DEVELOPMENT

Reduced protein kinase A (PKA) activity in anterior imaginal disc cells leads to cell-autonomous induction of decapentaplegic (dpp), wingless (wg), and patched (ptc) transcription that is independent of hedgehog (hh) gene activity. The resulting nonautonomous adult wing and leg pattern duplications are largely due to induced dpp and wg expression and resemble phenotypes elicited by ectopic hh expression. Inhibition of PKA in anterior cells close to the posterior compartment can substitute for hh activity to promote growth of imaginal discs, whereas overexpression of PKA can counteract transcriptional induction of ptc by hh in these cells. PKA therefore appears to be an integral component of the mechanism by which hh regulates the expression of key patterning molecules in imaginal discs.

Hedgehog stimulates maturation of Cubitus interruptus into a labile transcriptional activator

In Drosophila, signalling by the protein Hedgehog (Hh) alters the activity of the transcription factor Cubitus interruptus (Ci) by inhibiting the proteolysis of full-length Ci (Ci-155) to its shortened Ci-75 form,. Ci-75 is found largely in the nucleus and isthought to be a transcriptional repressor, whereas there is evidence to indicate that Ci-155 may be a transcriptional activator,,. However, Ci-155 is detected only in the cytoplasm, where it is associated with the protein kinase Fused (Fu), with Suppressor of Fused (Su(fu)), and with the microtubule-binding protein Costal-2 (refs 1,7,8,9). It is not clear how Ci-155 might become a nuclear activator. We show here that mutations in Su ( fu) cause an increase in the expression of Hh-target genes in a dose-dependent manner while simultaneously reducing Ci-155 concentration by some mechanism other than proteolysis to Ci-75. Conversely, eliminating Fu kinase activity reduces Hh-target gene expression while increasing Ci-155 concentration. We propose that Fu kinase activity is required for Hh to stimulate the maturation of Ci-155 into a short-lived nuclear transcriptional activator and that Su(fu) opposes this maturation step through a stoichiometric interaction with Ci-155.

Preferential expression in mushroom bodies of the catalytic subunit of protein kinase A and its role in learning and memory

Involvement of the cAMP cascade in Drosophila learning and memory is suggested by the aberrant behavioral phenotypes of the mutants dunce (cAMP phosphodiesterase) and rutabaga (adenylyl cyclase). Line DCO581, isolated via an enhancer detector screen for genes preferentially expressed in the mushroom bodies, contains a transposon in the first exon of the catalytic subunit gene (DCO) of protein kinase A (PKA). RNA in situ hybridization and immunohistochemistry show that DCO is preferentially expressed in the mushroom bodies. The DCO581 insertion and an independently isolated hypomorphic allele (DCOB10) each produce homozygous lethality and a 40% decrease in PKA activity in heterozygotes. This decrease has mild effects on learning but no effect on memory. However, the 80% reduction in activity obtained by constructing heteroallelic yet viable DCO581/DCOB10 animals results in a dramatic learning and memory deficit. These results suggest that PKA plays a crucial role in the cAMP cascade in mushroom bodies to mediate learning and memory processes.

Transducing the hedgehog signal.

Regardless of the status of Smo/Ptc associations, we now have to consider which, if any, of the newly found responses of Smo to Hh actually determine Smo signaling activity. As there really are no definitive data to guide us here, I will take the opportunity to discuss some possibilities. Although it is logical to expect that Smo activity would increase roughly in proportion to its total concentration, clear exceptions have already been noted in artificial situations where smo RNA is overproduced or PKA activity is inhibited. It is possible that in these situations, Smo protein does not reach the plasma membrane, or perhaps a particular domain of the plasma membrane, and that Smo concentration in a specific subcellular location is, in fact, the key determinant of Smo activity. However, even if this is true, additional regulation might be expected.One pivotal issue is that we do not know the biochemical activity of Smo. Although its seven transmembrane domain structure suggested that it might activate a trimeric G protein, there has been very little evidence supporting this idea prior to a recent study of human Smo in frog melanophores (DeCamp et al. 2000xDeCamp, D.L, Thompson, T.M, deSauvage, F.J, and Lerner, M.R. J. Biol. Chem. 2000; 275: 26322–26327Crossref | PubMed | Scopus (75)See all ReferencesDeCamp et al. 2000). Smo activation might nevertheless involve a conformational change that is characteristic of G protein coupled receptors (GPCRs). This idea comes from a recent study of the locations of amino acid substitutions that constitutively activate vertebrate Smo (Taipale et al. 2000xTaipale, J, Chen, J.K, Cooper, M.K, Wang, B, Mann, R.K, Milenkovic, L, Scott, M.P, and Beachy, P.A. Nature. 2000; 406: 1005–1009Crossref | PubMed | Scopus (847)See all ReferencesTaipale et al. 2000). One could easily imagine a conformational change resulting from binding of Hh to a Ptc–Smo complex or from Hh-induced dissociation of the complex. In the case of GPCRs, this creates a binding site for G proteins (Bockaert and Pin 1999xBockaert, J and Pin, J.P. EMBO J. 1999; 18: 1723–1729Crossref | PubMedSee all ReferencesBockaert and Pin 1999). In the case of Smo, this might create a binding site that links it directly to a cytoplasmic Ci effector complex.What about Smo phosphorylation? Although increased Smo phosphorylation might result in accumulation of active Smo at the plasma membrane (Denef et al. 2000xDenef, N, Neubuser, D, Perez, L, and Cohen, S.M. Cell. 2000; 102: 521–531Abstract | Full Text | Full Text PDF | PubMedSee all ReferencesDenef et al. 2000), such causative links have not been established. In reality, we might expect a more complex relationship that will require tracking the status and impact of each site of Smo phosphorylation. One reason for expecting complex regulation by phosphorylation is that we already know of two kinases, PKA and Fused, involved in Hh signaling; we know that several proteins (Fused, Costal-2, and now Smo) become hyperphosphorylated during Hh signaling (Ingham 1998xIngham, P.W. EMBO J. 1998; 17: 3505–3511Crossref | PubMed | Scopus (358)See all ReferencesIngham 1998), and yet, there is also some evidence that Hh signaling might reduce the phosphorylation of some proteins (Chen et al. 1999xChen, C.-H, von Kessler, D.P, Park, W, Wang, B, Ma, Y, and Beachy, P.A. Cell. 1999; 98: 305–316Abstract | Full Text | Full Text PDF | PubMed | Scopus (201)See all ReferencesChen et al. 1999). For the sake of discussion, I outline below one possible model for how specific kinases and phosphorylations might regulate Hh signaling at the cell surface.The role of PKA demands special attention because Alcedo et al. have shown that Smo protein accumulates dramatically when PKA is inhibited. There is no evidence yet regarding the relevant target for PKA, but Smo contains a cluster of putative PKA sites in its C-terminal cytoplasmic domain. It is therefore tempting to draw a direct analogy to similar sites in Ci and guess, as Alcedo et al. suggest, that PKA targets Smo for degradation by direct phosphorylation. If this occurs, Hh signaling could lead to the accumulation of hyperphosphorylated Smo simply by blocking degradation of PKA-phosphorylated Smo. To complete this model I would suggest that a Ptc-associated protein (perhaps a kinase) normally targets PKA-phosphorylated Smo for degradation (Figure 1DFigure 1D), and that Hh interrupts this process by dissociating Smo from Ptc. Hh/Ptc complexes would be internalized and degraded (Figure 2Figure 2), limiting the movement of Hh across cells (Chen and Struhl 1996xChen, Y and Struhl, G. Cell. 1996; 87: 553–563Abstract | Full Text | Full Text PDF | PubMed | Scopus (626)See all ReferencesChen and Struhl 1996) and prolonging the half-life of activated Smo roughly in proportion to Hh dose. Liberated Smo might bring one or more associated kinases to the Ci complex, leading to phosphorylation of Costal-2 and Fused. These phosphorylations may in turn stimulate release of Ci-155 from cytoplasmic anchorage, association of Ci-155 with a transcriptional coactivator and hence, movement of active Ci-155 into the nucleus. These last ideas are, of course, highly speculative and represent just one of many models that will be prompted by the recent reports delving into Smo and Ptc metabolism.*E-mail: ddk1@columbia.edu

Molecular cloning and predicted full‐length amino acid sequence of the type Iβ isozyme of cGMP‐dependent protein kinase from human placenta

In this study we report the isolation and characterization of three overlapping cDNA clones for the type Iβ isozyme of cGMP‐dependent protein kinase (cGK) from human placenta libraries. The composite sequence was 3740 nucleotides long and contained 58 nucleotides from the 5′‐noncoding region, an open reading frame of 2061 bases including the stop codon, and a 3′‐noncoding region of 1621 nucleotides. The predicted full‐length human type Iβ cGK protein contained 686 amino acids including the initiator methionine, and had an estimated molecular mass of 77 803 Da. On comparison to the published amino acid sequence of bovine lung Iα, human placenta Iβ cGK differed by only two amino acids in the carboxyl‐terminal region (amino acids 105–686). In contrast, the amino‐terminal region of the two proteins was markedly different (only 36% similarity), and human Iβ cGK was 16 amino acids longer. In a specific region in the amino‐terminus (amino acids 63–75), 12 out of 13 amino acids of the human Iβ cGK were identical to the partial amino acid sequence recently published for a new Iβ isoform of cGK from bovine aorta. Northern blot analysis demonstrated a human Iβ cGK mRNA, 7 kb in size, in human uterus and weakly in placenta. An mRNA of 7 kb was also observed in rat cerebellum, cerebrum, lung, kidney, and adrenal, whereas an mRNA doublet of 7.5 and 6.5 kb were observed in rat heart. Comparison of Northern and Western blot analyses demonstrated that the mRNA and protein for cerebellar cGK increased during the development of rats from 5 to 30 days old, whereas the 6.5 kb mRNA in rat heart declined.

Drosophila Smoothened phosphorylation sites essential for Hedgehog signal transduction

The Hedgehog (Hh) signalling pathway is crucial for animal development and is aberrantly activated in several types of cancer. In Drosophila melanogaster, Hh signalling regulates target gene expression through the transcription factor Cubitus interruptus (Ci). Together, Protein Kinase A, Casein Kinase 1 and Glycogen Synthase Kinase 3 silence the pathway in the absence of ligand by phosphorylating Ci at a defined cluster of sites, thereby promoting its proteolytic conversion to a transcriptional repressor (Ci-75). In the presence of Hh, Ci-155 is no longer converted to Ci-75 and its ability to activate transcription is potentiated. All Hh responses require the seven transmembrane domain protein Smoothened, which itself becomes hyperphosphorylated during Hh signalling. Here we show that a cluster of protein kinase A and protein kinase A-primed casein kinase 1 phosphorylation sites in Smoothened, similarly distributed to those regulating Ci, are essential for Smoothened to transduce a Hh signal and for normal regulation of Smoothened protein levels.

Similarities between the Hedgehog and Wnt signaling pathways

Hedgehog and Wnt proteins are signaling molecules that direct many aspects of metazoan development through signal transduction pathways that are just beginning to be understood. Recently, the common use of glycogen synthase kinase 3 and casein kinase 1 has been added to a growing list of straightforward similarities between Hedgehog and Wnt signaling pathways. These kinases silence both pathways by labeling a key transcription factor (Cubitus interruptus) or co-activator (beta-catenin) for proteolysis, and it is possible that reversal of these phosphorylation events is, in each case, central to pathway activation. This review compares the two pathways to explore whether our more extensive knowledge of Wnt pathways can be of predictive value for investigating Hedgehog signaling.

Altered circadian pacemaker functions and cyclic AMP rhythms in the drosophila learning mutant dunce

Neural circadian pacemakers can be reset by light, and the resetting mechanism may involve cyclic nucleotide second messengers. We have examined pacemaker resetting and free-running activity rhythms in Drosophila dunce (dnc) and DC0 mutants, which identify a cAMP specific phosphodiesterase and the catalytic subunit of cAMP-dependent protein kinase, respectively. dnc mutants exhibit augmented light-induced phase delays and shortened circadian periods, which indicate altered pacemaker function. Interestingly, however, light-induced phase advances are normal in dnc, suggesting a selective effect on one component of the pacemaker resetting response. Furthermore, we demonstrate the presence of circadian rhythms in cAMP content in head tissues and show that dnc mutations increase the amplitude of daily cAMP peaks. These results show that cAMP levels are not chronically elevated in the dnc mutant. A role for cAMP signaling in circadian processes is also suggested by an analysis of DC0 mutants, which have severe kinase deficits and display arrhythmic locomotor activity.